A genome-wide association scan reveals novel loci for facial traits of Koreans.

DNA-based prediction of externally visible characteristics (EVC) with SNPs is one of the research areas of interest in the forensic field. Based on a previous study performing GWAS on facial traits in a Korean population, herein, we present results stemming from GWA analysis with KoreanChip and novel genetic loci satisfying genome-wide significant level. We discovered a total of 20 signals and 12 loci were found to have novel associations with facial traits, including six loci located in intergenic regions and six loci located at UBE2O, HECTD2, CCDC108, TPK1, FCN2, and FRMPD1. Additionally, we performed a polygenic score analysis for 33 distance-related traits in facial phenotyping and determined genetic relationships between facial traits and SNPs using the GCTA program. The results of the current study offer an understanding of how facial morphology is influenced by complex genetic structures and provide insights into forensic investigation and population genetics.

Applicable Forensic Biomarker for Drowning Diagnosis: Extracellular Signal-Regulated Kinase 2 (ERK2).

Drowning is a common cause of accidental death worldwide, and it continues to be a serious public health problem. However, diagnosing drowning is a challenging task in forensic investigation because it is difficult to prove actual drowning and other submerged deaths with the autopsy techniques that are currently in use. Here, we show biomarkers that may be helpful for the diagnosis of drowning. We divided the experimental animals into four groups (drowning, postmortem submersion, hypoxia, and control) to evaluate the expression patterns of extracellular signal-regulated kinase 1/2 (ERK1/2). On gene expression analysis, only ERK2 was found to be significantly increased in the drowning groups compared to the other cases. In the immunoblot analysis, phosphorylated ERK2 (p-ERK2) was found to be upregulated in the drowning groups. Immunohistochemical staining also showed that p-ERK in alveolar cells revealed a granular pattern in the drowning groups. However, the expression pattern of ERK2 over time after drowning differed between the freshwater and seawater drowning groups. Taken together, these results indicate that ERK2 may be useful for distinguishing between drowning and postmortem submersion if the postmortem interval (PMI) of drowning is short. Conversely, if the PMI is long from the time that death occurs until the discovery of dead bodies, it is possibly more helpful for identifying between freshwater and seawater drowning.

Genetic variants of FGFR family associated with height, hypertension, and osteoporosis.

BACKGROUND: Hypertension and osteoporosis are the most common types of health problems. A recent study suggested that the fibroblast growth factor receptor-like protein 1 (FGFRL1) gene in giraffes is the most promising candidate gene that may have direct effects on both the skeleton and the cardiovascular system. AIM: Our study purposed to replicate the finding that the FGFR5 gene is related to giraffe-related characteristics (height, hypertension, and osteoporosis), and to assess the associations between genetic variants of the FGFR family and three phenotypes. SUBJECTS AND METHODS: An association study was performed to confirm the connections between hypertension, osteoporosis, and height and the FGFR family proteins (FGFR1 to FGFR5). RESULTS: We identified a total of 192 genetic variants in the FGFR family and found six SNVs in the FGFR2, FGFR3, and FGFR4 genes that were associated with two phenotypes simultaneously. Also, the FGFR family was found to be involved in calcium signalling, and three genetic variants of the FGFR3 gene showed significant signals in the pituitary and hypothalamus. CONCLUSION: Taken together, these findings suggest that FGFR genes are associated with hypertension, height, and osteoporosis. In particular, the present study highlights the FGFR3 gene, which influences two fundamental regulators of bone remodelling.

Auranofin promotes antibacterial effect of doripenem against carbapenem-resistant Acinetobacter baumannii.

AIMS: This study was performed to identify the potential for repurposing auranofin as an antibiotic adjuvant against carbapenemase-producing Acinetobacter baumannii. METHODS AND RESULTS: The clinically isolated A. baumannii strains used in this study were all resistant to carbapenems and harboured the blaOXA-23 gene. The synergistic effect of auranofin and doripenem against carbapenemase-producing A. baumannii was confirmed through checkerboard and growth kinetic analyses. This study also demonstrated the inhibitory effects of auranofin against A. baumannii biofilms. The anti-biofilm effects of auranofin were visualized by confocal laser scanning microscopy (CLSM). Furthermore, auranofin inhibited motility, one of the virulence factors. Additionally, the changes in the expression of carbapenemase-, biofilm- and efflux pump-related genes induced by auranofin were confirmed via quantitative polymerase chain reaction (qPCR). CONCLUSIONS: Our results demonstrated that auranofin has an antibacterial effect with doripenem and an inhibitory effect on several factors related to carbapenem resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that auranofin is a promising antibiotic adjuvant that can be used to prevent antibiotic resistance in carbapenem-resistant A. baumannii.

Anti-bacterial and Anti-biofilm Effects of Equol on Yersinia enterocolitica.

Yersinia enterocolitica has clinical significance due to its etiological role in yersiniosis and gastroenteritis. This study was designed to assess anti-bacterial and anti-biofilm effects of equol on Y. enterocolitica via phenotypic and genetic analyses. To determine its anti-bacterial activity, minimum inhibitory concentrations (MICs) of equol against clinically isolated Y. enterocolitica strains were analyzed. Subsequently, it was confirmed that the sub-MIC90 value of equol could inhibit biofilm formation and reduce preformed biofilm. Furthermore, it was found that equol could reduce the expression of biofilm-related (hmsT) gene in Y. enterocolitica. This study also demonstrated that equol not only reduced levels of bacterial motility, but also decreased the expression of a motility-related (flhDC) gene in Y. enterocolitica. XTT [2,3-bis (2-metoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction analysis revealed that equol attenuated cellular metabolic activities in Y. enterocolitica biofilm. Additionally, changes in biomass and cell density in equol-treated biofilms were visualized using a confocal laser scanning microscope. In conclusion, this study suggests that equol is a potential anti-bacterial and anti-biofilm agent to treat Y. enterocolitica.

The interaction between FTO rs9939609 and physical activity is associated with a 2-fold reduction in the risk of obesity in Korean population.

OBJECTIVE: FTO (fat mass and obesity-associated) gene is a well-known genetic risk factor for obesity. We investigated whether physical activity modulates the effect of FTO rs9939609 on obesity in Korean population. METHODS: The study analyzed the correlation between physical activity and obesity in 8840 individuals representing the Korea Association Resource (KARE). The association between obesity-related traits and single-nucleotide polymorphisms (SNPs) was assessed using linear regression models. Physical activity was defined as 3 hours or more of daily intense activity. RESULTS: Participants carrying rs9939609 (AT+AA) genotypes showed higher BMI compared with those carrying the wild-type (TT) homozygote. The highest significant association was observed between obesity-related traits (ß = .334, P value = 1.76 × 10-6 ). FTO rs9939609 (AT+AA) increased the risk of obesity (OR = 1.42, CI [1.13-1.79]), which was correlated with BMI correlations. However, active exercise by subjects carrying the same genotype reduced the risk of obesity by nearly 2-fold (OR = 0.62, CI [0.25-0.84]). In contrast, TT genotype was not statistically significant in reducing the risk of obesity in the active exercise group. CONCLUSIONS: Our results support a previous finding correlating FTO and obesity-related traits and suggest that the interaction with genetic variation and physical activity is an important risk factor for obesity.

Anti-biofilm and anti-virulence effects of zerumbone against Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that affects patients with a compromised immune system and is becoming increasingly important as a hospital-derived infection. This pathogen is difficult to treat owing to its intrinsic multidrug resistance and ability to form antimicrobial-tolerant biofilms. In the present study, we aimed to assess the potential use of zerumbone as a novel anti-biofilm and/or anti-virulence agent against A. baumannii. The results showed that zerumbone at sub-inhibitory doses decreased biofilm formation and disrupted established A. baumannii biofilms. The zerumbone-induced decrease in biofilm formation was dose-dependent based on the results of microtitre plate biofilm assays and confocal laser scanning microscopy. In addition, our data validated the anti-virulence efficacy of zerumbone, wherein it significantly interfered with the motility of A. baumannii. To support these phenotypic results, transcriptional analysis revealed that zerumbone downregulated the expression of biofilm- and virulence-associated genes (adeA, adeB, adeC and bap) in A. baumannii. Overall, our findings suggested that zerumbone might be a promising bioactive agent for the treatment of biofilm- and virulence-related infections caused by multidrug-resistant A. baumannii.

Antimicrobial and antibiofilm activities of Clostridium butyricum supernatant against Acinetobacter baumannii.

Acinetobacter baumannii is the major nosocomial pathogen that causes serious infections such as ventilator-associated pneumonia and bacteremia due to its biofilms. Hence, this study investigated the antimicrobial and antibiofilm potentials of cell-free supernatants (CFS) obtained from Clostridium butyricum, as probiotic, against A. baumannii. Our results demonstrated that C. butyricum CFS inhibited A. baumannii cell growth in planktonic culture. Also, C. butyricum CFS not only inhibited the biofilm development and dispersed mature biofilms, but also suppressed the metabolic activity of biofilm cells, showing antibiofilm activity. The biofilm components reduced by C. butyricum CFS were observed via confocal laser scanning microscopy. In addition, C. butyricum CFS exhibited antivirulence effect by inhibiting the motility of A. baumannii. Furthermore, C. butyricum CFS significantly downregulated the expression of efflux pump-related genes including adeA, adeB and adeC in A. baumannii. Our data demonstrate that C. butyricum CFS showed antimicrobial and antibiofilm effects on A. baumannii. These effects are closely associated with suppression of motility and efflux pump-related genes in A. baumannii. The findings suggest that C. butyricum CFS can be used as a new therapeutic alternative against biofilm-associated infection caused by multidrug-resistant A. baumannii.

Antibiofilm and antibacterial activities of repurposing auranofin against Bacteroides fragilis.

The aim of this study is to evaluate the antibiofilm and antibacterial effects of auranofin against WT-ETBF, rETBF, WT-NTBF and clinically isolated Bacteroides fragilis strains. The minimum inhibitory concentration and biofilm inhibitory concentration of 0.25 µg/ml for auranofin against B. fragilis were determined, and the biofilm eradication concentration was 1 µg/ml. At an auranofin concentration of 0.5 µg/ml, little cellular metabolic activity was found. Confocal laser scanning microcopy results confirmed the inhibition of biofilm by auranofin. The effects of auranofin on the outer membrane protein (ompA) gene and the RND-type efflux pump (bmeB3) gene were investigated using quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR results showed that treatment with auranofin significantly reduced the gene expression compared to controls without auranofin. These data indicate the applicability of auranofin as a repurposed drug due to its inhibitory effect against biofilm formation of B. fragilis. Therefore, our study demonstrates that auranofin, already approved for human use, is a promising drug that has strong antibiofilm and antibacterial activity against B. fragilis.

Association between non-Caucasian-specific ASCC1 gene polymorphism and osteoporosis and obesity in Korean postmenopausal women.

INTRODUCTION: Osteoporosis is a common disorder characterized by decreased bone mineral density (BMD). Interestingly, osteoporosis and obesity have several similar features, including a genetic predisposition and a common bone marrow stem cell. With aging, the composition of bone marrow shifts to adipocytes, osteoclast activity increases, and osteoblast function declines, resulting in osteoporosis. MATERIALS AND METHODS: We performed a genome-wide association study (GWAS) analysis with osteoporosis and body mass index (BMI) and did identify an association in 349 and 384 SNPs by filtering with the significant p values (p < 0.001) of BMI and osteoporosis, respectively. RESULTS: Only three of those SNPs were common (rs2326365, rs7097028, and rs11000205) between the SNPs significantly associated with BMI and/or osteoporosis in Korean Association REsource (KARE) females. Two of the three SNPs belonged to the ASCC1 gene and one to the FAM50B gene. We carried out a minor allele frequency (MAF) analysis of the rs7097028 and rs11000205 SNPs in the ASCC1 gene with a geographic genome variant browser. Both rs7097028 and rs11000205 in the ASCC1 gene were seen mostly in African and Southeast Asian populations. CONCLUSIONS: Our results suggest that the ASCC1 gene is a significant genetic factor for determining the risk for both osteoporosis and obesity in KARE postmenopausal females.

Enterotoxigenic Bacteroides fragilis infection exacerbates tumorigenesis in AOM/DSS mouse model.

The azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model is commonly used to study colitis-associated cancer. The human commensal bacterium, enterotoxigenic Bacteroides fragilis (ETBF) secretes the Bacteroides fragilis toxin (BFT) which is necessary and sufficient to cause colitis. We report that BALB/c mice infected with WT-ETBF and administered three cycles of AOM/DSS developed numerous, large-sized polyps predominantly in the colorectal region. In addition, AOM/DSS-treated BALB/c mice orally inoculated with wild-type nontoxigenic Bacteroides fragilis (WT-NTBF) overexpressing bft (rETBF) developed numerous polyps whereas mice infected with WT-NTBF overexpressing a biologically inactive bft (rNTBF) did not promote polyp formation. Unexpectedly, the combination of AOM+ETBF did not induce polyp formation whereas ETBF+DSS did induce polyp development in a subset of BALB/c mice. In conclusion, WT-ETBF promoted polyp development in AOM/DSS murine model with increased colitis in BALB/c mice. The model described herein provides an experimental platform for understanding ETBF-induced colonic tumorigenesis and studying colorectal cancer in wild-type mice.

Antibacterial and antibiofilm effects of α-humulene against Bacteroides fragilis.

The rapid increase in antibiotic resistance has prompted the discovery of drugs that reduce antibiotic resistance or new drugs that are an alternative to antibiotics. Plant extracts have health benefits and may also exhibit antibacterial and antibiofilm activities against pathogens. This study determined the antibacterial and antibiofilm effects of α-humulene extracted from plants against enterotoxigenic Bacteroides fragilis, which causes inflammatory bowel disease. The minimum inhibitory concentration and biofilm inhibitory concentration of α-humulene for B. fragilis were 2 µg/mL, and the biofilm eradication concentration was in the range of 8-32 µg/mL. The XTT reduction assay confirmed that the cellular metabolic activity in biofilm rarely occurred at the concentration of 8-16 µg/mL. In addition, biofilm inhibition by α-humulene was also detected via confocal laser scanning microcopy. Quantitative real-time polymerase chain reaction (qPCR) was also used to investigate the effect of α-humulene on the expression of resistance-nodulation-cell division type multidrug efflux pump genes (bmeB1 and bmeB3). According to the results of qPCR, α-humulene significantly reduced the expression of bmeB1 and bmeB3 genes. This study demonstrates the potential therapeutic application of α-humulene for inhibiting the growth of B. fragilis cells and biofilms, and it expands the knowledge about biofilm medicine.

Effects of fingerprint development reagents on subsequent DNA analysis.

A variety of evidences are found at crime scenes. Fingerprint and DNA evidences are especially important in the process of identifying personal sources. Among evidences found at crime scenes, cigarette butts are important because they might contain both fingerprints and DNA. In this study, latent fingerprints were detected in cigarette butts using 1,8-diazafluoren-9-one (DFO) and 1,2-Indanedione/zinc chloride (1,2-IND/Zn). Next, DNA extraction and real-time qPCR were performed to quantify and identify the DNA present. Short tandem repeat (STR) profiling was also performed. The results showed that the quantity of DNA recovered was decreased by 16% in DFO-treated cigarettes and by 27% in 1,2-IND/Zn-treated cigarettes when compared to untreated controls. When the STR profiling results were compared with those of the control sample, DFO, and 1,2-IND/Zn reagent-treated DNA samples showed individualized genotyping at several loci. Results of this study showed that when cigarette butts were found, DFO and 1,2-IND/Zn reagents could be used for DNA profiling after fingerprint identification. However, the effect of DFO on STR profiling was less than that of 1,2-IND/Zn. Therefore, we recommend the use of DFO for fingerprinting cigarette butts if further DNA processing is planned.

Efficacy of zerumbone against dual-species biofilms of Candida albicans and Staphylococcus aureus.

Candida albicans and Staphylococcus aureus are the most common opportunistic pathogens that co-exist as mixed biofilms. Dual-species biofilms of C. albicans and S. aureus cause nosocomial medical device-related infections that are strongly resistant to antibiotics and host immune responses compared with mono-species biofilms. The purpose of this study was to describe the efficacy of zerumbone derived from Zingiber zerumbet (L.) Smith, on dual-species biofilm formation. This study examined the inhibitory effects of zerumbone on planktonic cell growth, adhesion and biofilm formation. The results demonstrated that zerumbone remarkably inhibited mono- and dual-species biofilms formed by C. albicans and S. aureus using the XTT [2,3-bis(2-smethoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide]-reduction assay. Furthermore, a significant decrease in biomass and cell density of dual-species biofilms following zerumbone treatment was confirmed using confocal laser scanning microscopy (CLSM). Therefore, our study suggests that zerumbone is a potential antimicrobial and antibiofilm agent indicated for the therapeutic management of nosocomial medical device-related infections induced by dual-species biofilms of C. albicans and S. aureus.

Zerumbone inhibits Candida albicans biofilm formation and hyphal growth.

Candida albicans biofilm formation is considered an important matter because it can lead to strong resistance to conventional antifungal agents. Hyphae formed by C. albicans can also act as an important virulence factor related to its biofilm. The objective of this study was to determine the effect of zerumbone, a monocyclic sesquiterpene extracted from Zingiber zerumbet (L.) Smith, against C. albicans biofilm formation. Our results suggest that zerumbone possesses antifungal and antibiofilm activity that inhibits biofilm formation and eradicates preformed biofilm. Notably, zerumbone considerably reduced carbohydrate and DNA contents of biofilm matrix. In addition, zerumbone showed antivirulence effects by decreasing the growth of hyphae and inhibiting morphologic changes of C. albicans. Furthermore, zerumbone significantly downregulated expression levels of biofilm-related and hyphae-specific genes, including HWP1 and ALS3. Since zerumbone suppresses biofilm formation and hyphae growth, these results indicate that zerumbone could be used as a potential candidate to treat and prevent C. albicans biofilm-related infections.

Anti-biofilm and antimicrobial effects of zerumbone against Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis (ETBF) can form biofilms in the colon mucosal membrane and may promote chronic infection and tumor formation. The objective of this study was to determine the effect of zerumbone extracted from Zingiber zerumbet (L.) Smith, on B. fragilis biofilm formation. Inhibitory effects of zerumbone on planktonic cell growth and biofilm formation were examined by crystal-violet biofilm assays and XTT metabolic assays. Results showed that zerumbone significantly inhibited biofilm formation and eradicated established biofilms. Furthermore, B. fragilis biofilms inhibited by zerumbone were observed by confocal laser scanning microscopy. qRT-PCR was used to support the phenotypic results and to investigate the expression levels of an efflux pump-related gene (bmeB). Specifically, among the efflux pump-related genes, zerumbone significantly suppressed the expression level of bmeB12. These results indicate that zerumbone might be a promising candidate as an anti-biofilm and antimicrobial agent to treat and prevent biofilm-related infections caused by B. fragilis.

Anti-biofilm and Anti-Virulence Efficacy of Celastrol Against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a multi-drug resistant opportunistic pathogen that causes nosocomial infections in immunocompromised patients. This pathogen is difficult to treat owing to its intrinsic multidrug resistance and ability to form antimicrobial-tolerant biofilms. In the present study, we aimed to assess the potential use of celastrol as a novel anti-biofilm and/or anti-virulence agent against S. maltophilia. Results showed that celastrol at its sub-inhibitory doses decreased biofilm formation and disrupt the established biofilms produced by S. maltophilia. Celastrol-induced decrease in biofilm formation was dose-dependent based on the results of the microtiter plate biofilm assays and confocal laser scanning microscopy. In addition, our data validated the anti-virulence efficacy of celastrol, wherein it significantly interfered with the production of protease and motility of S. maltophilia. To support these phenotypic results, transcriptional analysis revealed that celastrol down-regulated the expression of biofilm- and virulence- associated genes (smeYZ, fsnR, and bfmAK) in S. maltophilia. Interestingly, celastrol significantly inhibited the expression of smeYZ gene, which encodes the resistance-nodulation-division (RND)-type efflux pump, SmeYZ. Overall, our findings suggested that celastrol might be a promising bioactive agent for treatment of biofilm- and virulence-related infections caused by the multi-drug resistant S. maltophilia.

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs.

The effectiveness of anti-biofilm and anti-virulence properties of dihydrocelastrol and dihydrocelastryl diacetate in fighting against methicillin-resistant Staphylococcus aureus.

Human pathogens have readily been converted into multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), because of the long-term use of conventional antibiotics. In addition, the biofilms formed by S. aureus cells are especially problematic and are related to the persistence of chronic infections because they constitute a major mechanism of promoting tolerance to diverse antimicrobial agents. Hence, the inhibitions of biofilm formation and/or toxin production are accepted as alternative means of controlling S. aureus infections. The present study was aimed at identifying novel anti-biofilm and/or anti-virulence compounds in friedelane-based pentacyclic triterpenoids present in many edible and medicinal plants-and investigating them against MRSA strains. As a result, dihydrocelastrol and dihydrocelastryl diacetate were found to both inhibit the biofilm formation of, and to disrupt the preformed biofilms of, MRSA strains to an increasingly greater degree with increasing concentrations of each compound. Furthermore, these two triterpenoids also clearly inhibited the hemolytic activity of MRSA-and in-line with their anti-biofilm activities, rendered the cell more hydrophilic. Additionally, corroborating phenotypic results, transcriptional analyses showed that both dihydrocelastrol and dihydrocelastryl diacetate disturbed the expression of gene related to α-hemolysin (hla) and down-regulated the expressions of the crucial biofilm-associated genes (agrA, sarA, ica, RNAIII, and rbf) in MRSA. The findings of this study suggest that friedelane-based pentacyclic triterpenoids-especially dihydrocelastrol and dihydrocelastryl diacetate-have the potential to be candidates both for use in controlling biofilm-related infections and for use as important components of anti-virulence strategies for fighting against MRSA infection.